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Microhomology-assisted scarless genome editing in human iPSCs

Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Ne...

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Autores principales: Kim, Shin-Il, Matsumoto, Tomoko, Kagawa, Harunobu, Nakamura, Michiko, Hirohata, Ryoko, Ueno, Ayano, Ohishi, Maki, Sakuma, Tetsushi, Soga, Tomoyoshi, Yamamoto, Takashi, Woltjen, Knut
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838097/
https://www.ncbi.nlm.nih.gov/pubmed/29507284
http://dx.doi.org/10.1038/s41467-018-03044-y
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author Kim, Shin-Il
Matsumoto, Tomoko
Kagawa, Harunobu
Nakamura, Michiko
Hirohata, Ryoko
Ueno, Ayano
Ohishi, Maki
Sakuma, Tetsushi
Soga, Tomoyoshi
Yamamoto, Takashi
Woltjen, Knut
author_facet Kim, Shin-Il
Matsumoto, Tomoko
Kagawa, Harunobu
Nakamura, Michiko
Hirohata, Ryoko
Ueno, Ayano
Ohishi, Maki
Sakuma, Tetsushi
Soga, Tomoyoshi
Yamamoto, Takashi
Woltjen, Knut
author_sort Kim, Shin-Il
collection PubMed
description Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes.
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spelling pubmed-58380972018-03-08 Microhomology-assisted scarless genome editing in human iPSCs Kim, Shin-Il Matsumoto, Tomoko Kagawa, Harunobu Nakamura, Michiko Hirohata, Ryoko Ueno, Ayano Ohishi, Maki Sakuma, Tetsushi Soga, Tomoyoshi Yamamoto, Takashi Woltjen, Knut Nat Commun Article Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes. Nature Publishing Group UK 2018-03-05 /pmc/articles/PMC5838097/ /pubmed/29507284 http://dx.doi.org/10.1038/s41467-018-03044-y Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Kim, Shin-Il
Matsumoto, Tomoko
Kagawa, Harunobu
Nakamura, Michiko
Hirohata, Ryoko
Ueno, Ayano
Ohishi, Maki
Sakuma, Tetsushi
Soga, Tomoyoshi
Yamamoto, Takashi
Woltjen, Knut
Microhomology-assisted scarless genome editing in human iPSCs
title Microhomology-assisted scarless genome editing in human iPSCs
title_full Microhomology-assisted scarless genome editing in human iPSCs
title_fullStr Microhomology-assisted scarless genome editing in human iPSCs
title_full_unstemmed Microhomology-assisted scarless genome editing in human iPSCs
title_short Microhomology-assisted scarless genome editing in human iPSCs
title_sort microhomology-assisted scarless genome editing in human ipscs
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838097/
https://www.ncbi.nlm.nih.gov/pubmed/29507284
http://dx.doi.org/10.1038/s41467-018-03044-y
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